Early detection of primary tumors is the key for effective therapeutic intervention and successful patient survival. Small animal models emulating human diseases are powerful tools for our comprehensive understanding of the pathophysiology of tumor formation and metastasis to distant sites. Our long-term goal is to develop a non-invasive, multiphoton-fluorescence lifetime imaging (MP-FLIM) modality that can precisely quantify these steps in animal tumor models at a very early stage. The working hypothesis is that fluorescence lifetime can be employed as reliable contrast parameter for providing higher detection sensitivity as compared with conventional intensity-based tumor imaging approaches and therefore it is possible to detect smaller tumor volumes (early detection) than those achieved by other prevailing methods. We base this hypothesis on our recent observations that (1) fluorescence lifetime is "intrinsic" to the fluorophore and its measurement is not affected by concentration and/or spectral artifacts as in intensity-based methods, (2) multiphoton excitation can enable increased tissue penetrability and reduced phototoxicity and (3) MP-FLIM approach can discriminate background autofluorescence from the fluorescent proteins in thick tissues thereby achieving a ten-fold increase in signal-to-background ratio over the intensity-based approaches. Based on these observations, we will focus on evaluating the efficacy of the synergetic combination of (multiphoton excitation + lifetime detection) in early detection of primary tumors in vivo. Specific Aim 1: To develop a MP-FLIM system and to characterize the system for multi-component lifetime analysis in small animal models by autofluorescence discrimination;The major goal of this specific aim is to develop a prototype MP-FLIM modality for three-dimensional imaging of early progression kinetics of primary tumors in vivo;Systematic characterization of light propagation in tissues and the combined effects of system optics and tissue properties on measured lifetimes will be evaluated and utilized in the design and fabrication of the in vivo imaging platform. Image analysis protocols will be developed for 3D image reconstruction, lifetime deconvolution and for correcting tissue scattering/absorption effects. Specific Aim 2: To test the efficacy of MP-FLIM system in imaging early stages of primary tumors in breast tumor xenograft models and in monitoring gene therapeutic response in tumors in vivo;the major goal of this specific aim is to generate primary tumors in nu/nu mice and to test various aspects of early stage tumorigenesis. Multicomponent lifetime analysis will be employed to discriminate tumor cell fluorescence from normal autofluorescence background. Three-dimensional quantification of tumor burden will be carried out. FLIM-based tumor pH imaging (microenvironment) and NADH imaging (redox metabolism) will be carried out. Finally the recently developed nonlinear dynamical analysis of glucose metabolism in tumors will be evaluated for its diagnostic potential in vivo. Significant outcome of this project will be the development of an innovative, non-invasive tumor imaging modality for monitoring early stages of tumor progression in intact animal. Future Projections: By the end of year 2, we will have setup the prototype, multimodal MP-FLIM system for small animal imaging and will have extensively characterized the efficacy of lifetime detection in monitoring of early stages of primary tumor growth invivo. In collaboration with our clinical collaborators at Cedars-Sinai Medical center, we are keen in taking the present prototype system to the next level for clinical diagnosis and gene therapeutic monitoring. Our future projections for the multimodality, MP-FLIM system in clinical setting are as follows: v Monitor efficacy of Gene-therapeutic agents in small animal models;v Utilize the present MP-FLIM preclinical platform as a learning tool to develop a clinical diagnosis system;Adapt the protype platform for optical-fiber based laser delivery/detection system;v Extend the scope of the microscope platform to endoscopic applications;v Develop a portable, clinical FLIM-imaging modality;Implement an intra-operative system for real-time assessment of Glioblastoma multiforme. PUBLIC HEALTH RELEVANCE This exploratory proposal by a new investigator aims to develop a novel multiphoton fluorescence lifetime imaging system with a focus on non-invasive, early detection of primary tumors invivo in mouse models. These findings will pave a way for monitoring the real-time effects of pharmacological intervention on reducing tumor burden in vivo.